Anti-tubercular agents



United States Patent 955cc ANTI-TUBERCULAR AGENTS Peter P. T. Sah, Davis, Calir., assignor to The Regents of the University of California, Berkeley, Calif.

No Drawing. Filed Sept. 28, 1953,'Ser. No. 382,838

6 Claims. (Cl. 167-65) This invention relates to new chemical compositions that not only possess a high degree of bactericidal effectiveness against micro-organisms, but which are further characterized by a remarkably low degree of toxicity. More particularly, this invention relates to a means and to chemical compositions useful for combating, preventing the spread, and, under proper conditions, eliminating tuberculosis in human beings and other animals.

For years men of science and medicine have sought a means to combat the contagious disease caused by the tubercle bacillus (Mycobacterium tuberculosis). The disease is exceedingly widespread, and it has been estimated that tuberculosis causes about one-eighth of all deaths. The seriousness of the disease in humans depends on the organ involved, the virulence of the strain of tubercle bacilli and the resistance of the individual infected. Almost any portion of the body may be involved in the tubercular process, although the commonest site is the lungs. As a result, the commonest method of transmit-ting the human strain is through droplet infection sprayed into the air by coughing or the drying of tubercular sputum which is spread around as dust. The bacilli may live in the dry sputum for years, and in the dust form they are distributed over wide areas. In cities nearly everyone is constantly breathing in live tubercle bacilli.

Until recently, the treatment of the disease was based almost solely on the recuperative powers of the body aided by proper therapy such as fresh air, diet, bed rest, collapse treatment, and good nursing. Several years of strict regime are necessary for arr sting or curing the disease,. and relapses are common even after apparent cure.-

Recently, significant advances have been made in the treatment of human tuberculosis due to the discovery of certain drugs, such as streptomycin, which act directly to inhibit the growth or mulL'plication of the bacillus. In this way these drugs help the recuperative powers of thebody to dispose of the disease more effectively and in much less time. More recently, isonico-tinic acid hydrazide, a known compound, was found to have similar antitubercular properties. This fact is disclosed in United States Patent No. 2,596,069. Other compounds also known to have antitubercular activity are thio-semi-carbazone, and para-amino-salicylic acid. However, each of the above named drugs have, in common, certain significant shortcomings:

(1). They are toxic so that in useful doses the drugs.

arenot always well tolerated;

(2). When the drugs are given in tolerable doses, the.

drugs have only a bacteriostatic rather than a bactericidal activity towards the tubercle bacilli, that is they merely inhibit the growth of the bacilli rather than destroy it;

(3 T he drugs have had little or no eifect on advanced fibrotic type cases, particularly thoseoflong duration, andzhave been more or less limited in their use to a fresh exudative type of the disease; and

(4) The bacilli may learn to live with the drug so that new or resistant strains are developed which are not affected by the drug at all. These resistant strains of bacilli may spread to other patients who, as a result, will be unable to respond to even initial treatment with the drug. This problem particularly characterized the development of streptomycin therapy, and now is a factor in the evalu-f ation of any new chemotherapeutic agent.

additional problems of its own in use.

vertigo, and dizziness.

binations of certain drugs, such as streptomycin and paraamino-salicylic acid, have been used to delay the appear-- ance of resistant strains of bacilli, but never with perma-* nent success.

In particular, study of isonicotinic acid hydrazidehas'f indicated a high degree of toxicity in humans undoubtedly due partially to the presence of the free hydrazino group. For example, it has been reported that isonicotinic acid' hydrazide, given in large doses, may produce the follow-.

ing side effects: blood dyscrasia, anemia, constipation, and

difficulty of urination. Consequently, the maximumsafe dosage of this drug for human use has been 250' to 300 mg. per day or less. Such doses are not bactericidal, but

are only suihcient to inhibit the growth of the bacilli." There may also be a marked stimulation of the central nervous system, evidenced by convulsions or respiratory" trouble, which is therapeutically undesirable, particularly in the treatment of tuberculosis where complete rest and quiet are need. (J. Amer. College of Chest Phys., vol.

XXXIH, No. 1, 1953, Robitzek et al., pp. 8-1-0, Wi-tkind et al., pp. 21-25; Dela State Med. 1., August 1952,

Flaherty, pp. 198, 199.) In addition, rains of tubercle bacilli resistant to isonicotinic acid hydrazide rapidly dc Likewise, the existence of resistant strains of. bacilli not previously responsive to the drug have also been reported. (Antibiotics and Chemotherapy, vol. 2,

velop.

No.9, pp. 484-486.)

The present invention is directed to new chemical com positions which have a high degree of bactericidal activity against micro-organisms yet which are comparatively much less toxic than compounds'presently used in treat-1' Conse quently, one object of the present invention is to produce} new isonicotinyl hydrazines from the d-uronic acids, their salts, and their lactones which are not only bactericidal in'f their activity but which also are suiliciently non-toxicto permit their full use in utilizing this bactericidal activity ing diseases caused by such microorganisms.

against Mycobacterium tuberculosis.

Another object of the present invention isto produce new isonicotinyl hydrazones which act as chemical moie'-' ties to achieve higher blood concentrations of an anti tubercular agent than is possible with the free isonicotinic acid hydrazide.

Another object is to produce new compounds for'com-f bating human tuberculosis that are so much less toxic than other known active compounds for this purpose as I to permit their administration in bactericidal dosages.

no appreciable efiect in stimulating the central nervous system when used'as a human medicament.

Another object is to produce bactericidal compounds: that will act to diminish lung lesions and the volume of sputum resulting from tuberculosis in a human patient,-

Patented June 14, 1960 dosages of antitubercular resistant strains.

- andwhich willalso render such sputum harmless due to the absence of live bacteria.

Another object is to produce an active antitubercular agent that will produce no harmful side efiect in the patient or animal to whom it is administered 'Many other objects and advantages of the present invention will appear from the following description. As has been noted, isonicotinic acid hydrazide is known to be a very active antitubercular agent but cannot be satisfactorily used as such due to its extreme toxicity and its tendency to stimulate the central nervous system. The present invention takes advantage of this activity bycondensing the hydrazide with the d-uronic acids aridtheir. lactones, which are known detoxicating agents, to produce new ISOIllCOllDYl hydrazones having a chemically stable bond between the hydrazide and the carbonyl groups. These new compounds have exhibited excellent antitubercular characteristics when tested in vitro, in vivo, and clinically. Not only have these drugs demonstrated a] marked activity against Mycqbacterizim tuberculosis, but the fact that the drugs have proved to be somuch less toxic to humans than is the free hydrazide, the uncombined isonicotinic acid hydrazide, has permitted mit complete destruction of the bacilli in animals as well as'in human patients. 7 Because of prior research, including work on the deriva- I tives of isonicotinicacid'hydrazide, these favorable results could not be readily foreseen. In' general, one combining a non-polar biologically active substance with a polar, detoxicating substance, such as the uronic acid derivatives, would expect the biological properties to be lost by'the combination. Instead, it has been found that the activity of the uronic acid derived isonicotinyl hydrazones on an equal weight basis is approximately equal to that of the fiee isonicotinic acid hydrazide; while the toxicity of these new drugs is approximately 1/ 10th that of the uncombined hydrazide. As a consequence, the new isonicotinyl hydrazones'have been ad ministered to patients in amounts up to 2,500 to 3,000 mg. per day with no untoward efiects.

: From a clinical standpoint, these drugshave resulted in a noticeable decrease in the size and number of tubercular lesions in patients lungs, and a corresponding re duction in the volume ofsputu'm produced. 'Also the sputum thatis produced is negative, that is, it no longer contains the tubercle bacilli. 7

Of equal or even greater significance at highldosage, no strains of tuberculosis bacilli have been able to live with the drug long enough to form drug The probable reason. for this is that blood level concentrations correspond to the high drug dosage possible with the new hydrazones,,thereby per mitting complete elimination of the tubercle bacilli and indicating no drug resistanceat these, concentrations.

Of even more significance is the fact that comparative testshave shown that bacteria strains resistant to the action of isonicotinic acid hydrazide have been attacked and destroyed by equal concentrations of the new hydrazones, indicating a protective capacity of the new drugs in an area where the free hydrazide is ineffectual. In addition, thehydrazones formed fromthe d-uronic acids and their lactones have shown no measurable tendency'to stimulate the'central nervous system, or to cause blood dyscrasias or other side eflects in patients tested, a, very important therapeutical factor in the treatment of humans. Based on a comparison with the uncombined hydrazide, these facts indicate that the new isonicotinyl hydrazones act as a moiety, and not by hydrolysis to produce the free hydrazide; I

Broadly, the new isonicotinyl hydrazones are prepared by dissolving the d-uronic acid derivative in'a suitable solvent, such as methyl alcohol, and adding the isonicotinic: acid hydrazide. The reactants are then boiled'a suflicient time to allow the condensationTreaction to occur.

the new drugs in suflicient ,quantitiesto per-- The reaction mixture is then c W led to room temperature with the isonicotinyl hydrazones appearing as crystals within a relatively short time, i.e. 24 hours or less. The crystals are filtered from the solution and, preferably, are washed and finally dried by suction.

The following are illustrative examples of the preparation of three representative isonicotinyl hydrazones; using -1 procedures that resulted in very high yields of the crystalline products obtained. a r

One of-the isonicotinyl hydrazones prepared from a lactone is d-glucuronolactone isonicotinyl hydrazone,

OH H OH OH a three liter round bottom flask, was added 1.5 liters of is the fact that,

methyl alcohol (acetone-free). The mixture was boiled gently on a steam bath for 10 minutes, producing a clear solution. To this hot solution, 70 grams of isonicotinic acid hydrazide was added all at once. The mixture was a then boiled vigorously for 10 minutes and the clear solution filtered Without suction through a piece of'lens paper into a two liter Erlenmeyer flask. After the flask had been allowed to stand 24 hours at room temperature,

crystals in the form of beautiful white rods and narrow plates were observed. .These crystals'were filtered'with suction, washed with a 'small'amount of methyl alcohol,

and sucked to complete dryness. The resulting product was dried in a vacuum desiccator for three days. Actual yield was 148 grams, or a percentage yield of better than 99%. i

0n heating the d-glucuronolactone isonicotinyl hydrazone thus formed, the crystals charred and decomposed with foaming between 150 and C. without any sharp melting point. The particular decomposition point depended on the rate of heating and the type of apparatus used. The new'compound was readily soluble in' water from'which it could not be recrystallized. It was practically' insoluble in cold methyl or ethyl alcohol but slightly soluble in'boiling solutions of these solvents (100 cc. of methyl alcohol dissolved about"1.2 grams ofthev product at 66 C.)." The new compound was already very pure, and upon recrystallization from a large amount of methyl or ethyl alcohol (absolute) showed noappreciable change in physical properties from the unrecrystallized product. 1

EXAMPLE 2 D-glucuronolactone isonicotinyl hydrazone has also been prepared from sodium d-glucuronate as follows: v D-glucuronic acid Was'liberated from a solution of its sodium salt'(12.0 g.) in water (25 ml.) by the addition of concentrated hydrochloric acid '(5 ml). To the mixture, sodium acetate (5 g.) was added to remove the excess of the mineral acid. Isonicotinic acid hydrazide (7.0 g.) was then introduced into the clear solution and mixing accomplished by thorough shaking. Methyl alcohol (250 ml.) was added and the mixture boiled on a steam bath for 10 minutes. 7 A white crystalline precipitate started to separate from the solution after a few minutes heating and was allowed to stand overnight at room temperature before filtering. The crystals (white small rods) were suclged to dryness, washed with a small amount of absolute methanol and again sucked to dryness. The product was further dried in a vacuum desicca'tor for twenty-four'hours. Yield: 25-50 percent; The hydrazone melted at '150 -160 and decomposition. 2.

screens 5 EXAMPLE 3 An isonicotinyl hydrazohe prepared 'from a uronic also a new compound that is highly active against Mycobacterium tuberculosis and comparatively much less toxic than isonicotinic acid hydrazide. This compound has been prepared as follows:

In an Erlenmeyer fiaskof one liter capacity, 16.7 grams of isonicotinic acid hydrazide was dissolved in 170 cc.

of methyl alcohol (acetone-free) by heating on the steam bath. To the hot solution 25 grams of d-galacturonic acid, dissolved in 150 cc. of hot distilled water, was added. The reactants were thoroughly mixed by shaking and the flask heated on the steam bath for 10 minutes. Crystalline precipitate (white platelets) was seen to form even when the solution was boiling hot After twenty-four hours of standing at room temperature, the

crystalline product was filtered off with suction, washed first with a small amount of ice-cold distilled water, and

then with a small amount of methyl alcohol, and finally sucked to dryness. The product was placed in a vacuum desiccator of anhydrous CaCl where it was dried for three days. Theweight of the resultant crystalline prodnot was 40.5 grams (92% of the theoretical yield). n heating, it charred and decomposed between 212 and 214 C.

In contrast to d-glucuronolactone isonicotinyl hydrazone, which could not be recrystallized from an aqueous medium, d-galacturonic acid isonicotinyl hydrazone was readily purified by recrystallization from boiling water in the presence of a small amount of decolorizing charcoal. From analytical data, however, it is apparent that the original product is almost as pure as the recrystallized. There is no appreciable difierence in physical properties. The new compound is also freely soluble in dilute NaHCO NaOl-I, and HCl. In sodium bicarbonate solution it may be used for injection in animals either intravenously or intraperitoneally.

EXAMPLE 4 D-galacturonic acid isonicotinyl hydrazone has also been prepared as :follows, using water as the solvent.

D-galacturonic acid (100 g.) was dissolved in distilled (500 1111.). Isonicotinic acid 'hydrazide (65 g.) was also dissolved, separately, in the same solvent (250 ml.) by warming on a steam bath. The hot d-galacturonic acid solution was filtered and the clear solution added to the isonicotinic acid hydrazide solution. The mixture was thoroughly mixed by shaking and then heated on the steam bath for 10 minutes. White crystals started to separate even while the solution was still hot. The mixture was allowed to stand for twenty-four hours at room temperature. The crystals (white plates and rods) were filtered with suction, washed with a small amount of ice-cold distilled water, and then with some methanol. Suction was continued until the product became dry. The product was further dried in a vacuum desiccator over anhydrous calcium chloride for twentyfour hours. Weight: 124 g. The hydrazone melted at 207-208 (uncorn); 211-212" (corn) with chairing and decomposition. 7

EXAMPLE Another isonicotinyl hydrazone prepared from a uronic acid derived lactone is d-mannuronolactone isonicotinyl hydrazonc,

6 also a new compound with co paratively low toxicity and very 'higha'ntitube'rcular activity. This compound has been prepared'in the following manner:

On a steam bath, 17.5 grams of d-mannuronolacto'ne boiled with .a mixture of 350 cc. of methanol and 150 cc. of distilled water until a clear solution resulted. To this solution 14 grams ofisonicotinic acid hydraz'ide was added all at once, and the mixture boiled continually for ten minutes on a steam bath. The resulting clear solution was filtered, while hot, through a piece of lens paper into a 500 cc. Erlenmeyer flask and allowed to stand. After twenty-four hours, crystals in the form of fine white needles were observed and were filtered oli with suction, washed with a small amount of ice-cold methyl alcohol, and sucked to dryness. The product was further dried in a vacuum desiccator for twenty-four hours. The total yield was 24.8 grams of theoretical yield).

The pure crystals of d-mannuronolactone isonicotinyl hydrazone melted between 150 to 175 C. with foaming and decomposition. This compound may also be further purified by recrystallization from 75% methanol.

It'is a'notable feature of the three new. isonicotinyl hydrazones, just described, that each may be producedfrom a uronic acid or lactone that is readily available and easily manufactured. In particular, dsgalacturonic acid may be prepared from Waste products such as orange or other citrus fruit peels. Of the lactones', d-mannuronolactone is prepared by hydrolysisof alginic acid from seaweed; while d-glucuronolactone may be produced from glucose, particularly by-product glucose from starch manufacture. It is significant that the latter is also a metabolite, e.g., a composition normally occurring in warm-blooded animals.

The new isonicotiny-l hydrazones have been found to be highly active against tuberculosis in vivo and in vitro as well as clinically, while retaining a very low degree of toxicity. In tuberculosis infection of mice, in vivo tests indicated that 80 to percent protection could be provided the animals by feeding them a diet containing only 0.02 to 0.05 percent of the new isonicotinyl hydrazones. On the other hand, in comparative toxicity tests of mice the LD 50 dose for the new hydrazones fell generally within the range of; 1200 to 1800 mg./kg., while the LD 50 dose for the free hydrazide was m-g./kg. These results indicate that, generally speaking, equal weights of the new hydrazones are as active as the free hydrazide but only $5 as toxic. Additional indications based on the compounds tested, are that no appreciable diiference exists in either the activity or toxic effects of the various isonicotinyl hydrazones derived from the uronic acids and their lactones.

in vitro activity is also marked. Tests of the new ism nicotinyl hydrazones have indicated an ability to completely inhibit the growth of mycobacterium tuberculosis at concentrations of at least 0.2 microgram/cc. of culture or less, and, where the dilution was made, at concentrations of 0.02 microgram/cc. or less.

The antitubercular efficacy of d-glucuronolactone isonicotinyl hydrazone was also compared to that of isonicotinic acid hydrazide (both alone and in combination with streptomycin) in tests upon guinea pigs. These tests indicated again, that this new compound was at least as active as the free hydrazide, and revealed a capacity of the new drug to heal tuberculosis lesions by resolution at indicated dose levels. Corresponding doses of the :uncombined isonicotinic acid hydrazide produced similar results. It is noted that these tests do not reflect toxic effects of the drugs since animals generally have a comparatively much higher drug tolerance than humans. Significant, however, is the indication that sufiiciently large doses of the new drugs may result in complete destruction of the tubercle bacilli.

Acute and chronic toxicity tests were also performe -o'n'dogs to determine the efiect of the new compounds on tinyl hydrazone, corresponding to a drug consumption of ibodilyfunctions. At no time during these te'sts was any 1.15 mg. per mouse per day, I n H damage to. the organs orany significant flmctionalchange This test was conducted fora period of .2 8 days, the dbserired;'""when'compared to'the reportedeffectsv of results of which are tabulated in Table II. 9 isonicotinic acid hydrazide in similar tests, e.g. muscular 16 1 Y I s w tremblings or convulsions, salivation, mydriasis, loss j w Table H ,1 of weight, etc., these tests iurtherindicatethat the new w isonicotinyl hydrazones are comparatively much less toxic W l 7N0, I Mean than thc'free hydrazide; f v a r .i Compound 1 P ercent1n of Percent urviv'al The antitubercular activity and relatively low toxicity ;10 j 1 Diet l? M Tag? of the new isonicotinyl hydrazones upon animals is $110 in the f11wing examplfis- In a Pi P d-galachironic acid isonico o.023; '10 28+ ments, except those performed on dogs, a smgle strain of tinyl hydrszone. A V 7 animal bred under uniform conditions of nutrition and 1 FP FP P f- 9 2 3165+ environment were'used. V cente I g 90 183+ EXAMPLE6 r ii" 'F' A group of 30 mice averaging 18 to -s in thefanimals were sacfified on the28th day and weight, were each iniected'intravenously .with.0.4 cc. of to i d, A in, none of the: {ed ,on diets a human strain ofmycoba'cterium tuberculosis (H37R V) 2O m j j d.- 1g m i id i i ti l hydrazqne ata'concentration of 2.5 mg./'cc. in Youmans medium o any of the surviving streptomycin animals showed having 10%v soluble starch added. Commencing .24 any gross tuberculous lesions in the lungs; whi1e,.al1 the hours after injection of the crganismaten'of'the animals l a i ls sh0we d widespread tuberculousglesions we'rel-'placed on av plain unmedicatedicommcrcially i 'th l g 1 uavailalblemouse diet, as :COHt'I'OIS...CFOI comparison, ten *2 EXAMPLEB' ammalswere also in ected, subcutaneously. w1th 3000 V, saventyrtwo Emails" can bigsguach weighing- 600 t grams) were inoculated .substernally with 0.0001 .g 'am .Fiveanimals wereallowcdtojeed adlibitum 'from the mentisnam rclebagfl'h (VI-80) They ,sarne mousediet. containing 0.05 percent-ofrpowde'red d- 'glucuronolactone; isonicotinyl; hydrazone. l Based on. the ..;observation1that mice normally consume .5 .0 grams of r ioodlper day, this .dictcorrespondS to a drug consumption of mg. per mouse per day. i r 7 Five animals were alsov placed on' the same mouse diet containing 0.02 percent of po'wered deglucuronolactone ,isonicotinyl hydrazone, corresponding'to "1.0 'mg: per

mouseperlday. .7 r

30 weregdivided into; 6 r numbered gr upw 1 animals each with twelve guinea pigs in group 1 beinginsedj as controls, The others were left nntreatedlforll days. -Oneanimal ingroup 4 died duringthe twenty-onegday qpcriod from pulmonary embolism; and-one control animal' wassacrificednforobservationat the end of the -period'and was found to have generalized tuberculosis. The. remaining animals in'the 6 groups received'daily for seven days, respectively: H V

t The test'period was 20 days. The results of these tests (1) No medicationv V p, V V V V are tabulated in Table I: 1 40 '(2) 1 mg. of isoni tinic acid hydrazide intramuscu- V flarly 1 ,j r '1 (3) 10 mg. of isonicotinic acid hydrazide intramuscularly M (4) 10 mg. of isonicotinic acid hydrazide plus 6 mg. Page ,32 of streptomycinintramuscularly 7 M c Mortality 53; 7 .(5) 10 mg'. of d-glucuronolactone isonicotinyl hydra- T Compound Percent in Diet z onebyintubation' a (6) 25 mg. of d-glucuronolactoneisonicotinyl hydrad-gldouronolhetone 7 isonic o- 0 7 20+ 'ttin lh drszona; 002-- 0 1 18,2 zone by intubation; Streptomycin" 0. .204. e

. v g By 39 days (6,0 days after beginningthc experiment) Control .10 I 7 four v of thefcontrolanimals had died," and 'had 'lostlan 1 average of 50 grams in weight. I {The "guinea pigs ,were sacrificed. The average .weighf. ;'gain .forfthe I7 Di h 20th 'day all the animals were sacrificed and 55, Surviving n-' 1 animals was 9 g m M 'autopsled'; 9? T l animals 'f For all-animals receiving isonicotinic hidhydrazide 70H diets contammg lsonlcotlllyl the average increase in weightwas-flfljgramsf "For all hydrazone'or strqptomycm Showed any gross'tub'erculous types of d g1ucuronolactone isonicfltinyl hydrazon clesions in theillmgsl the 'COIIU'OI animals, howwer, treated animals the average increase was 300 grams.

I showed generalized tuberculosis, evidenced in' each case Necropsy l d. generalized tuber ulosis f in all the y Wlder Spread tuberculous leslonscontrol animals. 7 In the 1 mg. isonicotiniciacid hydrazide Th?se F m confirmed Pther f accurate guinea pigs evidence of active tuberculous infection was experiments in which the above mdlcated daily doses of Vgnqtevd in the lungs f twogarnimalrs The stmptomycin plus d'glllcul'ollolactofle lsonlcoilnyl hydl'azone were admin" isonicotinic acid hydraz ide animals showed no evidence istered by intravenous injection into the dorsal vein in 55 f tuba-6111953 but one Small tubercle, whip}; appeared m 1 f reach file m l' l g fh W f have healed was noted in the lung of one animal. Si1 nilar results were achieved with the 10 mg. isonicotinic acid I g EXAMPLE 7 g ydrazide group 'A'group of 30 mice were infected intravenously in the Y In all the animals receiving .thenew hydra'zonethere same manner as described in Eiraniple 6. Again, 'ten was no gross-or microscopic evidence'of any tuberculous animals were placed on' unmedicated mouse diet as coninfection, except for one small tubercle in the 25 mg. ';trol's, and ten animals injectedsubcutaneouslyfoncei a treated group, ,and the animals appeared, generally, to sweek, with 3000units oi streptomycin sulfatesl'l hexrehave beencuredbyresglution u Q. a

maining 10 animals .werejfed rad b t-llm .mOUSGadifi These results were eanfifmea aTsiniilar tegbn other zsontaininsfi-pzire sent qv r a a mron c;as dzismi -fl guinea niaiin su a t tuberculosis attests EXAMPLE9 In a comparative test, percent Solutions of each of the following compounds wereprepared by dissolving in sterile distilled water:

(1) D-glucuronolacto'ne isonicotinyl hydrazone (2) D-galacturonic acid iso'nicotinyl hydrazone (3) D-marmuronolactone isonicotinyl hydrazone, and

(4) 'Isomcotinic acidhydrazide Mice, weighing 20 to 25 grams in'weight, were divided into groups of 20, and several groups were injected intraperitoneally with varying doses of one of the solutions. This procedure was repeated'until 'varying doses of each of the compounds had been administered to a number of groups, with 20 mice being used' at a single dose. The mortalities at each dose were observed fite'r four to five hours, and the results were plotted graphically for each dose of each compound.

The interpolated dose necessary to kill half of the animals in a group (LD 50) "was then determined from the plot of each compound. The results are tabulatedin Table III.

Table III Compound LD 60 mgJlrg.

lsonicotinic acid hydrazide 150 d-glucuronolactone isonicotinyl hydrazone. 1, 200 .d-galacturonic acid isonicotlnyl hydrazone.-. 1, 800 d-mannuronolactone isonicotinyl hydrazone.-. 1, 500

These results indicate that, generally speaking, the new compounds are'only $5 as toxic "as the free hydrazide.

The acute and chronic toxicity of the new'compounds in tests on dogs is illustrated in the following examples:

tion of sodium pentabarbital. The -'carotid "artery was cannulated and connected to a "mercury-manometer arranged to record the blood-pressure on 'a kymograph.

A pneumograph was placed aroun'dthe chest and connected to a tambour to record respiratory movement on the kymograph. A rubber balloon'was'inserted through an incision into the small intestine and then connected to a tambour to record intestinal movements on the kymograph. After a recording of normal values, the drug to be testedwas injected into the femoral vein and a continuous recording of respiration, blood pressure,

pulse rate and intestinal motility was-made over a period of an hour.

The following drugs were tested by the above procedure, giving in each case, a dose of 100 mg./k-g. of:

The results in every case were the same in that at no time during or following the injection of the compounds was there a significant change in respiration, pulse rate, blood pressure or intestinal motility.

EXAMPLE 11 To determine chronic toxicity in dogs, four healthy on a diet of "Skippy" ddg foodfor a week. Two of these dogs were then given daily doses of 50"mg.'/kg.'of"the"dwere sacrificed using sodium pentabarbital and autopsied by a pathologist. The following is a list of tests performed on the dogs:

(a) BLOOD EXAMINATION Red cell count, hemoglobin reticulocyte count, sedimentation time, white cellcount, diiierential count in white cells, non-protein nitrogen, blood sugar, prothrombin.

a (b) URINE EXAMINATION Albumen, casts, sugar, bile, bacteria,white cells, red cells, crystals,'epithelial cells. v

When compared. to the findings of these tests before starting the drugs, there was no significant change in-any of them with the exception of'the red count, hemoglobin and prothrombin which showed a definite improvement. There wasnothing found at autopsy in any of the organs to indicate 'any damage or'functional'change. These-results indicate that in the doses given, notoxic eifect can be demonstrated on liver function, kidney function, or

blood formation.

The activities of the new isonicotinyl hydrazones in vitro are shown in the following examples.

EXAMPLE-12 Dilutions of d-glucuronolactone isonicotinyl hydrazone in Youmans medium were prepared having final concentrations of 0.2, 0.02, 0.002, and 0.0002 mg./cc., and510 cc. of each were placed into separate test tubes. A fifth test tube of Youmans medium was prepared as a control. Each test tube was inoculated with 0.2 'cc. of a 7 day old culture of Mycobacterium tubercul0sis (H37RV) in Youmans solution, modified by the addition of 0.01% Tween 80. This inoculation corresponds to approximately 0.2 mg. of cell material per tube. A ten day eriod of incubation at 37 'C. was allowed, and then cell suspensions were prepared by autoclaving thetubes at 121 C. under 15 pounds, pressure for 45 minutes. The tubes were then adjusted to a volume of 10 cc. and read in a Coleman Spectrophotometer at 660 Mu. The results of these readings are tabulated in Table IV.

Table r12 N I Percent Trans- It will be observed from these results that -d-glucuronolactone isonicotinyl hydrazone achieved a complete inhibition of the growth of Mycobacierium tuberculosis down to a concentration of 0,2 microgram/ cc. Dilutions below this concentration were not made or tested indicating further investigation may be desirable, particularly in View of the results obtained with d-mannuronolactohe isonicotinyl hydrazone (Example 14).

EXAMPLE 13 Experiments were carried out in the same way as in Example 2 using identicaldilutions of d-galacturonic acid isonicotinyl hydrazone in Youmans solution, theresults of which are tabulated in Table V.

These results indicate that d-galacturonic acid isonicotinyl hydra zone may also accomplish almost complete inhibition ofthe. growth ,of Mycobacterium tuberculosis at concentrations at least as small as 0.0002 mg./ cc.

. EXAMPLE 14 a D-nnannuronolactone isonicotinyl hydrazone was tested for antitubercular, activityin vitro, using the method described by Rakeet al. (Am. Rev. Tub., vol. 60, No. '1, July 1949). The BCG strain of the bovine type tubercle bacillus was grown in Kirchner Synthetic Medium for the "seed culture. The assay medium was of a' modified Kirch- "'n'er formula containing 2 cc. of seed'culture per 100 cc.

' Dfas'saymedium; The compound was'used at two-fold dilutions in testtubes inoculated with the bacilliso that 'the first tube"contain'ed"0.6 mg./cc.', while, the final dilution contained 0.0025 microgram/cc; Tests were in- ,m actual studies, doses up to timesthe -tolerated amount of isonicotinic acid hydrazide have been administered without sults confirm the harmful effects. These unexpected recal moieties, rather than by hydrolysis decomposition in the blood stream, which, thereby, achieve greatly reduced 1 toxicity because of the new chemical combination.

' Following the favorable conclusion of tests in vitro and on animals, several clinical studies were undertaken to evaluate the obvious advantages inherent in the reduced toxicity of the new drugs. v r a a One clinical studyof eleven patients is reported in the following example; It. is significant that a majority of these cases were cases of long duration, including several advanced types which are generally classified as hopeless.

-The example is coneerned with the first group of patients who completed three or more months of treatment, and

indicates recovery in seven of thecases and marked im- 7; short time of a, fewmonths.

'c'ubated at -37'C. fo'r 112 hour'sl- Results-we're based on gmrbidity (growth) at'the end of the incubation period.

At the end of thetest period the tests were compared with;

' 'two sets of coutrols', one inoculated with the bacilli and the other containing only the Kirchner medium; The inoculated control wasfvery turbid while the control mediumwaswater clear. By this test it was determined Y that d mannuronolactone isonicotinyl "hydraz'one had, a"

inhibiting concentration atf 0.02 microgram/cc. ;(dilution of 1:50,000,000), atwhich concentration no evidence of growth was observed. i V 'fThc following example demonstrates the antitubercular activity of d-glucuronolactone isonicotinyl hydrazone against 18 strains of Mycobaeteriuni tuberculosis which had shown previous resistance to the action of the -free 'hydrazide.

Eighteen plates'w'e're" prepared 'each' containing 12 cc. of'a solution having a concentration of 10 micrograms *of d-glucuronolactone isonicotinyl hydrazoneper cc. These plates were then inoculated each with 0.5 cc. of the fserums of patients known to be infected with LEXAMPLEIS- These results are all the more in the relatively provement in tworothers remarkable since they'were achieved EXAMPLE l6 Eleven tuberculosis patients who had never beentreated with isonicotinic acid hydrazideand whose sputum was constantly positiveto direct tar-concentrated smears were selected on a voluntary basis for clinical. trial. All patients were either moderately or far advanced cases J of whichfoun were.fibro-caseous typesrofllong. duration,

months to inexcess ofgtwenty years.

three were old cases having large cavities, one involved a bronchogenic fistula, and three were fresh exudative type cases." -Known"duration of disease varied from six An initial dose of 60Qjn1g. per day of d-glucuronolactone isonicotinyl hydrazone was administered orally in tablet form. Every few days this was increased approximately 50 percent,

- until a maximumdaily dosage of 2,400 mg. was reached. .No untoward etfects .wereobserved at this high dosage level, although treatment was discontinued in one patient due to polyneuritis while the daily dosage was reduced from 2,400 mg. to 1,800'n'1g. inanother. The treatment was continued in the remaining patients for three or more months before the present data was accumulated, in-

- dicating that a daily dose of 2,400 mg. is probably not excessive in the average case. Pneumoperitoneum was given simultaneously in two cases, and was begun after 6 to 8 weeksin three others. o

Mycobacterium tuberculosis which, based on previous identical tests conductedon the same serums, were known to be more than 50% -resistant'to'theaction' of equivalent amounts of isonicotinic acid hydrazider After an incubation period of more than four weeks only two of the lates Shbwed' any'si ns of 'g'rowth';whereas, 16 ofthe Yplates proved negative on-testing and were incapable jot producing subsequent eaaefiaigrewth; l l. n Additional investigations have been 'carried outrwhich a i hjt a results, 3 u 6l 4if ejP v Q examples, and, with particular reference to d-glucuronolactoneisonicotinyl hydrazone, have indicated that equal amounts of the new drugs are as active asanyconcentrations 'of isonicotinic -acid hydrazide ever used against Mycobaoter ium tuberculosis. This is so even though the" comparative weights of molecular isonicotinic acid'hy- 'drazide in the respective drugs are entirely disproportiona te'. j Thus the theoretical amount of molecular "isonicotinic acid hydranide in d-glucuronolactone isonicotinyl hydrazone is only 41% that of an equal weight of the,

free hydrazide. Similar comparisons can be made as l-to .d -galacturonic acid-isonicotinyl hydrazone and dimannuronolactone isonicotinyl hydrazone; These 'facts alone mma aaica asne dosage levels-of the new drugs All ten of they patients continuing treatment'claimed to feel better, and those patients who were underweight invariably showed satisfactory weight gains; The cough was generally'calmed withina few weeks, and the expectoration' of sputum either ceased. or was, considerably reduced, in volume, More remarkable wasjthe rapidity with which the sputum was rendered negative. Sputum conversion occurred in 9 of the 10 treated patients within 5 months after starting the full dose of 2,400 mg. Sputum conversion occurred in 6' ofthesepatients, including the patient on whom treatment was stopped,

within one month after the start" of full'dosageq X-ray improvement occurred in 7 of the ten patients after only 3 months, andwas moderate to markedin 6. of these cases. In the casesnot showing X-ray improvement the known duration of diseasewas several years and their X-rays had been stable for variable'periods' never less than 6 or 8 months. 4 v p t The overall results indicated that 9" of the 10 treated patients were well on the way to recovery after only three 4 months while the patient failing to show sputum converindicatedin a range of=i1200'to--1800 nrgfpei:

sion nevertheless showed mild ,X-ray' improvement. Seven of the patients may be said to have recovered. It

appears justified to state, also; that the drug was relatively well tolerated at the high dosage level used, although chmcally efiective dosage levels have subsequently been 7 day, at whieh levels no compl ations dcvelopedi :1 p

belief that the new drugs act as chemincident;

-that high concentrations of anti-tubercular drug in the blood stream are desired for the treatment of certain typesof tuberculosis, especially military and meningeal. Reported blood levels for isonicotinic acid hydrazide are approximately 1.5 to 2.4 micrograms per cc. at the peak level which occurs about two hours after administration of 'a'near maximum dose of 300 mg. On the other hand, clinical studies of d-glucuronolactone isonicotinyl hydrazone have shown blood levels in the range of at least 12.5 to 24 micrograms per cc. for a similar dose, or approximately times that of the free hydrazide. Since low toxicities of the new hydrazones permit many times comparative dose, even higher blood levels are indicated at increased doses of the new drugs than are possible with isonicotinic acid hydrazide. I

The following example demonstrates blood level consentences for the d-glucuronola'ctone compound.

EXAMPLE 17 Blood levels were run on 16 patients, including the 11 specified in Example 18, with a total of 18 determinations being made. The blood levels were determined essentially as in theprocedure proposed by Rubin et a1. (Dis. Chest, volume 21, page 439, 19 52). The blood levels found ranged between 12.5 and 24 micrograms per cc. of plasma two hours after ingestion of a single dose of 330 mg. of d-glucuronolactone isonicotinyl hydrazone.

Of importance therapeutically is the ease with which the new drugs may be administered. Because of their crystalline form, the new isonicotinyl hydrazones may be easily compressed into tablets for oral use; or they may take other dosage forms suitable for oral ingestion, such as capsules or powders. Likewise, the solubility of the isonicotinyl hydrazones in harmless solvents such as distilled or sterile water, as well as their ease of assimilation into the blood stream, readily adapt them for parenteral use whether injected intravenously, subcutaneously, or intraperitoneally.

The low toxicity of the new hydrazones also simplifies the preparation of suitable dosage forms, and in most cases it is preferable to have the active material constitute the major portion of the dose. The following formulations, as well as the animal diets described in examples above, are purely illustrative of the many conceivable ways in which the new drugs may be administered, and are not intended to be in any sense limiting. For the sake of brevity the examples relate only to the d-glucuronolactone form of the new isonicotinyl hydrazones, but it will be apparent to one skilled in the art that similar formulations, such as the non-toxic salts thereof, would be suitable for any of the uronic acid derived isonicotinyl hydrazones.

Some examples of d-glucuronolactone tablet compositions suitable for oral administration are illustrated in Examples 18, 19, and 20.

EXAMPLE 18 Mg. D-glucuronolactone isonicotinyl hydrazone 200 Lactose-starch granules 100 Magnesium stearate 10 The ingredients were slugged, then granulated and retableted.

Conventional granulation methods were used.

are-as follows:

14 EXAMPLE 20 Mg. D-glucuronolactone isonicotinyl hydrazone 200 Corn starch 30 Glucose 50% Sufiicient quantity Calcium stearate 10 Some examples of elixirs suitable for oral ingestion nxAMrLE 21 D-glucuronolactone isonicotinyl hydrazone g 4 Propylene glycol i .2 i 46 Syrup U.S.P. cc

EXAMPLEZZ D-glucuronolactone isonicotinyl hydrazone g.. 4 Propylene glycol g 26 Syrup U.S.P. a g 30 Distilled water cc 100 Flavor and color Suflicient quantity One tablespoon of the elixir .is computed to be the equivalent of a 600 to 650 milligram dose of d-glucuronolactone in each of Examples 21 and 22.

A suitable method for preparing alarge lot of an injectable 'form of 'd-glucuronolactone isonicotinyl hydrazone is as follows:

i EXAMPLE 23 Eight liters of distilled water for injection, U.S.P., are placed in'a ZO-liter Pyrex glass bottle. To this water 500 grams of d-g'lucuronolac'tone isonicotinyl hydrazone are added with stirring until solution is effected. The pH is adjusted thereafter with suificient reagent grade N/10 hydrochloric acid to achieve a pH of about 5.0. Then suflicient water for injection is added to bring the volume to 10 liters. The solution is filtered and filled into clean, dry, sterile, flint glass ampuls, each containing 2.2 cc. of the fluid. The ampuls are sealed and sterilized for 30 minutes at F.

Such composition may crystallize under certain circumstances, but the crystals will be readily dissolved when the contents of the vial are heated to body temperature (37 C.).

It will be apparent to those skilled in the art to which this invention relates that widely diifering procedures, embodiments and applications of the invention, as well as some variation in the spatial relationships of the chemical structures, will suggest themselves Without departing from the spirit and scope of the invention. The non-toxic salts are one such embodiment. The disclosures and examples herein are purely illustrative and are not intended to be in any sense limiting. For example, optical isomers, such as the l-uronic acids and their lactones, as well as the racemates of such isomers, may subsequently prove to be as useful, or even more useful, than the d-uronic-compounds. Nor is there any intention to limit the use of the new compound solely to combating human strains of the tubercle bacillus, since the new drugs are thought to be effective against all forms of human tuberculosis whether caused by the human strain or the bovine (cattle) strain of the bacillus. Also, there is reason to believe that the new compounds may be efiective as therapeutic agents in the treatment of other acid fast organisms, such as mycobacterium leprae, and against micro-organisms responsible for some psychoses and certain dermatological manifestations, to indicate only a few additional uses.

The essential feature of this invention, however, is the production of new isonicotinyl hydrazones and their salts from uronic acids and their lactones which have substantial antitubercular activity, yet which are comparatively much less toxic than the free hydrazide drug, isonicotinic acid hydrazide. Stated cautiously, this activity, on an equal weight basis, is approximately equal to that of the free the free hydrazide.

larger dosage of thelnew. isonicotinylhydrazones than V a has ever been possible with the free hydrazide since at least ten times the'antitubercular activity in diseased hydrazone'of the formula:

hydrazide, while the .toxicityisnot more than that of This feature permits a considerably patients may be achieved without any further increase in harmful side efiects. In fact, the prospect of such toxic side. e'fiect is greatly reduced, since conversely, doses having equal activity would have but 5 the toxicity.

Actual dosage wi]l,-'of course, fallsomewhere in between. V

Whatisclaimedis:

1. "A new compound selected from the group consisting of d-glucuronolactone isonicotinyl hydrazone, d-galacturonic acid'isonico'tinyl hydrazon', d-mann'uronolactone V isonicotinyl hydrazone; andjthejnon-toxic salts thereof.

2. The new compound, d-galacturonic acid isonicotinyl 3; The new compound, d mannUronOlactone isonicotinyl hydraz'one. of the formulae: i V 7 inf-1 1 H fiiiifOfifo fi V p O 4. The method of treating tuberculosis infection without causing toxic side efiects in the infected organism which comprises administering to said organism a mini- 'adncumstenng to the patient a mlmmum dose of approxiv mately 600 mg. per day of a compound selected from the mum. dose of approximately 600 mg. per day of a compound selected from the group consisting of d-glucuronm lactone 'isonicotinyl hydraz one, d-gala'cturonie acid iso- ,nicotinyl hydra'zone d-mannuronolactone isonicotinyl hydrazone, and thenon-toxic salts thereof."

5 The new compound, lucuronolactone isonicotinyl v hydra'zone of the formula: w

H OH" H- i. v 7 6. The method of. treating tuberculosis in the human body without causing harmful side effects comprising group consisting ofd-glucuronolactone isonicotinyl hydrazone, d-galacturonic acid isonicotinyl hydrazone, d-man- 2o nuronolactone isonicotmyl hydrazone, and the non-toxic Selikofi: Quarterly Bull. Sea View I-Iosp Vol.13, No. 1, pp. 17-19. V f

& M UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent Ne 2,940,899 June 14 1960 Peter P, Ta Sah It is hereby certified that error appears in the-printed specification of the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column .2 line 49, for "hydrazines'" read hydrazones column 5, line 49, before "(500 1111,)" insert water column 13, line 5, for "military" read miliary Signed and sealed this 6th day of December 1960.,

(SEAL) Attest:

KARL Ha AXLINE ROBERT C. WATSON Attesting Oflicer Commissioner of Patents 

1. A NEW COMPOUND SELECTED FROM THE GROUP CONSISTING OF D-GLUCURONOLACTONE ISONICOTINYL HYDRAZONE, D-GALACTURONIC ACID ISONICOTINYL HYDRAZONE, D-MANNURONOLACTONE ISONICOTINYL HYDRAZONE, AND THE NON-TOXIC SALTS THEREOF.
 6. THE METHOD OF TREATING TUBERCULOSIS IN THE HUMAN BODY WITHOUT CAUSING HARMFUL SIDE EFFECTS COMPRISING ADMINISTERING TO THE PATIENT A MINIUM DOSE OF APPROXIMATELY 600 MG. PER DAY OF A COMPOUND SELECTED FROM THE GROUP CONSISTING OF D-GLURONOLACTONE ISONICOTINYL HYDRAZONE, D-GALACTURONIC ACID ISONICOTINYL HYDRAZONE, D-MANNURONOLACTONE ISONICOTINYL HYDRAZONE, AND THE NON-TOXIC SALTS THEREOF. 